Investigation On The Effects And Mechanisms Of Ceftazidime And Cefoperazone At Sub-MICs On Biofilm Formation Of Escherichia Coli Isolates

Background and ObjectivesEscherichia coli(E coli) is a commonly encountered pathogen that is often responsible for community- and hospital-acquired infections,and is the most familiar gram-negative bacteria. Treatment with multidrug-resistant E coli is very difficult. There are many reasons responsible for this difficulty, in which biofilm is very important.Biofilm,a common cause for persist bacterial infection,refers to bacteria that attach to surfaces aggregating in a hydrated polymeric matrix of their own synthesi s. It’s reported that there are approximately 80% bacterial infections which were associated with biofilm. Except for a physical barrier provided by bacteria biofilms, down-regulated bacterial growth and metabolism, and decreased expression levels of related genes and proteins, may help bacteria to inactivate the targets of antimicrobials and host immune system, and thus evade the attacks from antibiotics and host immune system. At the same time, through the dissociation of biofilm, bacteria may colonize in the new environment and grow there, leading to infection diffusion. Thus, biofilm makes antimicrobial therapy much more difficult. Seeking for new therapeutic schedule targeting at biofilm is now an emergent issue to be settled.In recent years, more and more investigations focus on the effects of antibiotics at sub-minimal inhibitory concentrations(sub-MIC). As we all knew, antibiotics at sub-MIC don’t interfere the growth of bacteria, while recent studies have shown that several antimicrobials at sub-MICs can affect some biological behaviors, including toxicity, athleticism and biofilm formation. This modulation may affect the drug treatment outcomes. In view of that sub-MIC is inevitable in the course of antimicrobial therapy, and at the same time, due to the increase of MIC in drug-resistant bacteria, time intervals for antibiotics at sub-MICs are even longer. Therefore, regulation of sub-MIC antimicrobial agents against biofilm should be deliberate to reveal bacterial resistant mechanism and to devel op novel antimicrobial therapy method.Mechanisms for antimicrobials at sub-MICs to affect bacterial biofilm formation are not clear. Response of Quorum-sensing system to antimicrobial drugs is likely to be one mechanism. Almost all kinds of bacteria have at least one quorum-sensing system. Activation of the quorum-sensing system may be induced by specific signaling moleculesnamed autoinducers. Therefore, regulation of auto-inducers is a central component of quorum-sensing system, and also may become main targets of drugs interference with QS. Sub-MIC antibiotics may inhibit the synthesis of bacterial QS auto-inducers and then reduce their virulence factors and biofilm production. This effect has been confirmed in some drugs and some strains. Researches on development of new antibiotics are much slower than resistance transmission. Therefore, screening in available drugs for potential capacity of controlling biofilm formation, and studying their mechanisms are significative for explorations of bacterial resistance mechanisms and development of new and effective treatment.In this research, we first evaluated the biofilm-forming ability of E coli clinical strains. Then selected two biofilm-producing E coli laboratory strains ATCC700926 and DH5α to observe the effects of antibacterial drugs at sub-MICs on their biofilm-forming ability. Results showed that Ceftazidime at sub-MICs may be able to inhibit the biofilm formation, and we then measured the effects of four common third-generation Cephalosporins on biofilm formation of 63 E coli isolates to screen the active drugs with potential capacity on biofilm formation. And then, we selected four isolates to observe the correlation of biofilm-modulating effects and drug resistance, evaluated the stability of the drugs, and measured changes in biofilm regulating genes. In the fourth part, we studied mechanisms of CAZ and CFP at sub-MICs on E coli biofilm formation via luxS/AI-2 quorum sensing.Methods1. Effects of third-generation Cephalosporins on biofilm formations by E coli isolates.1.1 Drug sensitive test:The minimum inhibitory concentration(MIC) of antibacterial agents on E coli was tested by the agr double dilution methods according to “Performance Standards for Antimicrobial Susceptibility Testing(2010 Edition)” published by Clinical and Laboratory Standards Institute(CLSI).1.2 Biofilm formation assay1.2.1 Determination for biofilm formationBiofilm formation was assayed by crystal violet staining of adherent cells. After incubation at 37 °C for 6–24 h, the plates were rinsed twice with phosphate-buffered saline and dried in an inverted position. The adherent cells were stained with 1% crystal violet for 10 min, and the dye was dissolved in 30% acetic acid, and the absorbance of the solubilized dye at 590 nm and bacterial density by absorbance at 600 nm were then determined.1.2.2 Dependence of the biofilm formation by E coli on the condition of cultureE coli ATCC700926 was selected in this experiment. Two species medium(LB medium and M9 medium), and three initial density of bacteria(106/L, and 107/L, and 108/L), and four incubating time(6h, and 12 h, and 18 h, and 24h) were employed. Biofilm-forming abilities by E coli isolates were evaluated by this formula.BF=(AB-CW) ?G.1.2.3 Evaluation of biofilm formation by E coliBiofilm formation by 63 E coli isolates were evaluated. The methods mentioned in 1.2.2 were used. Crystal violet staining of adherent cells on 96-well plates were employed. Incubating time was 24 h. Simultaneous determination of bacterial growth by Spectrophotometric method for determination at OD600 nm was used.1.3 Correlation analysis of biofilm formation and ESBLs1.3.1 Detection of the presence of ESBLsDouble-disk diffusion was used to detect ESBL production. The Cefotaxime(CTX) and Ceftazidime(CAZ) disks in combination with Clavulanate(CLA) were performed and interpreted by CLSI criteria for ESBL screening and disk confirmation tests. K. pneumoniae ATCC 700603 and E. coli ATCC 25922 were used as positive and negative controls, respectively.1.3.2 Correlation analysis of biofilm formation and ESBLsChi square test on the relativity of the biofilm formation and the presence of ESBLs were used to evaluate whether the ability of biofilm formation by E coli were correlated with the presence of ESBLs.1.4 Correlation analysis of biofilm formation and ESBLs genes1.4.1 Molecular detection of ESBLAll the isolates were screened by PCR using the specific primers for each couple primer. The PCR products were sent for DNA sequencing and the sequence results were analyzed using the NCBI BLAST program(http://www.ncbi.nlm.nih.gov/).1.4.2 Correlation analysis of biofilm formation and ESBLs genesChi square test on the relativity of the biofilm formation and the presence of ESBLs genes were used to evaluate whether the ability of biofilm formation by E coli were correlated with the presence of ESBLs genes.1.5 MLST analysisMultilocus sequence typing(MLST) was performed for the E. coli isolates. The primers of seven housekeeping genes, including adk, fum C, gyr B, icd, mdh, purA and recA, were PCR-amplified, purified and sequenced. The sequences were then compared with the MLST database(http://mlst.warwick.ac.uk/mlst/dbs/Ecoli), and each unique combination of alleles(the allelic profile) was designated as a sequence type(ST)2. Effects of antibacterial agents on biofilm formations by E coli isolates.2.1 Effects of nine antibacterial agents on biofilm formations by E coli ATCC700926 and DH5α.E coli strains ATCC700926 and DH5α were used in this evaluation. Nine antibacterial agents(CXM、CAZ、FEP、PIP、AMI、TOB、CIP、ERY、TE) at 1/4 MIC were evaluated. Crystal violet staining of adherent cells on 96-well plates were employed. Incubating time was 24 h.2.2 Effects of CAZ and other drugs on biofilm formations by E coli isolatesE coli strains with biofilm-forming abilities selected from experiments 1.2.2 were used in this evaluation. There are five groups: control(without medication) group and the drugs treatment groups(with 1/4 MIC of CAZ, CTR, CTX, or CFP). Crystal violet staining of adherent cells on 96-well plates was employed. Incubating time was 24 h.2.3 Dose-effects and time-effects relationship of CAZ and CFP on biofilm formationATCC700926, E46, E49 and E42 were chosen in this study. There were 11 groups in dose-effects analysis: control group(without drugs), CAZ 1/32-1/2MICs, and CFP 1/32-1/2MICs. Incubating times were 18 h. There were 12 groups in time- effects analysis: control(without drugs)- 6h, CAZ-6h, CFP-6h, controll-12h(without drugs), CAZ-12 h, CFP-12 h, controll-18h(without drugs), CAZ-18 h, CFP-18 h, controll-24h(without drugs), CAZ-24 h and CFP-24 h. Drug concentrations were 1/4MICs.3. Mechanism of CAZ and CFP on E coli biofilm formation3.1 Correlation test of sensibility of bacteria to CAZ and CFP and the effects of CAZ and CFP3.1.1 Correlation test of sensibility of bacteria to CAZ and CFP and the effects of CAZ and CFPChi square test on the relativity of the biofilm modulating activities of CAZ and CFP and the drug resistance to CAZ and CFP were used to evaluate whether the effects of CAZ and CFP at sub-MICs on E coli biofilm formation were correlated with the drug resistance of bacteria.3.1.2 Correlation test of ESBLs molecular and the effects of CAZ and CFPATCC700926、E46、E49、E42 were evaluated, and molecular detection of ESBL were carried as 1.4.1. 3.2 Concentration analysis of CAZ and CFP on biofilm formation of E coliHigh performance liquid chromatography(HPLC) was employed to analyze the change of the concentration of CAZ and CFP in the modulation of E46 and E11 during their biofilm formation.3.3 Measurement of mRNA changes of the genes encoding biofilm-modulating proteinsReverse transcription-polymerase chain reaction(RT-PCR) was used to investigate the levels of the mRNA products of the biofilm-modulating genes of this isolate with the treatment of CAZ and CFP.4. Mechanism of CAZ and CFP on E coli biofilm formation via lux S/AI-24.1 Influence of CAZ and CFP at 1/4 MICs on growth of E coli.Spectrophotometric detection was used for bacterial growth determination. For growth curve experiments, 50 μL of the culture sample was obtained every 4 h for 24 h to measure the optical density at 600 nm.4.2 Measurement of mRNA changes of lux SReal-time RT-PCR(q RT-PCR) was used to investigate the levels of the mRNA products of lux S of the four E coli isolates with the treatment of CAZ and CFP.4.3 AI-2 bioassay to determine the effects of CAZ and CFP at 1/4 MICs on AI-2 production4.3.1 Method for AI-2 bioassayV. harveyi BB170 was used as the reporter to determine AI-2 production. Dilutor factors of 1:500 and 1:5000 were examined, and incubating time of 1-6h were also evaluated.4.3.2 AI-2 bioassay to determine the effects of CAZ and CFP at 1/4 MICs on AI-2 productionThere were three groups(control, CAZ and CFP) for every isolates. The bioassays were performed in accordance with the method described in 4.3.1. For each experiment, the bioassay was performed in triplicate for each sample.4.4 Measurement of mRNA changes of the genes modulated by AI-2RT-PCR was used to investigate the levels of the mRNA products of the genes modulated by AI-2 of the four isolates with the treatment of CAZ and CFP.4.5 Transduction of anti-sense oligonucleotides(as-ODNs)We at first designed four as-ODNs(available at https://rnaidesigner.invitrogen. com/rnaiexpress/) and selected one with the best blocking efficiency for subsequent experiments. Briefly, 4 μL of the transfection reagent Sofast was diluted into 20 μL of LB broth. After incubation at room temperature for 20 min, 20 μL of as-ODN was added to the diluted transfection reagent. Forty microliters of the mixture was added to 160 μL of the competent bacterial suspension with a final concentration of 10 μM as-ODN. After culturing at 37 °C for 1 h, the transfected bacteria were adjusted to a turbidity equivalent of a 0.5 Mac Farlane standard suspension, and cultivated at 37 °C with shaking in an environmental chamber for 4 h. The AI-2 bioassay was performed to certify whether gene expression had been blocked. The effects of CAZ and CFP on biofilm formation of transfected bacteria were then determined.4.6 Effects of CAZ at 1/4MIC on biofilm formation by ATCC700926△lux SE coli strains ATCC700926△luxS was used in this evaluation. CAZ at 1/4 MIC were evaluated. Crystal violet staining of adherent cells on 96-well plates was employed. Incubating time was 24 h.The main results:1. Effects of third-generation Cephalosporins on biofilm formations by E coli isolates.1.1 Drug sensitive testMore than half of E coli isolates were resistant to penicillins, fluoroquinolones and cotrimoxazole. However, the strains responded better to the enzyme inhibitor and β-lactam antibiotics. The strains were highly resistant to Cefuroxime, while sensitive to CAZ and Cefepime. Only one strain was resistant to imipenem.1.2 Evaluation of biofilm formation by E coli isolates1.2.1 Dependence of the biofilm formation by E coli on the condition of cultureBiofilm may be formed by ATCC700926 in both culture mediums(LB and M9), while the strongest formation occurred in LB, and maximal amounts of biofilm may be obtained when incubating duration is 24 h, and initial bacterial density is 108 CFU/mL.1.2.2 Evaluation of biofilm formation by E coli isolates(AB-CW)/G was used as mathematical formula to quantify bacterial bio?lm by E coli clinical isolates. Results showed that almost all E coli clinical isolates(60/63) are able to form biofilm, in which 81% isolates were strong or moderate biofilm producers.1.3 Correlation analysis of biofilm formation and ESBLsIn the 63 isolates evaluated, there were 20 ESBL-producing E coli isolates. Chi square test on the relativity of the biofilm formation and ESBL-positive or not, confirmed that abilities of biofilm formation by E coli isolates are not correlated with their ESBL-producing.1.4 Correlation analysis of biofilm formation and ESBLs genesTwenty ESBL-producing E coli isolates were evaluated. Chi square test on the relativity of the biofilm formation and ESBL molecules, confirmed that abilities of biofilm formation by E coli isolates were not correlated with CTX-M, TEM, OXA and SHV.1.5 MLST analysisMLST results showed that 63 isolated strains had 31 gene types. The ST131 and ST167 were the most common genotypes with 10 and 7 strains separately. ST38, ST10 were less common with 4 isolates separately.2.1 Effects of nine antibacterial agents on biofilm formations by E coli ATCC700926 and DH5α.CAZ at 1/4MIC may inhibit biofilm formation by ATCC700926. Other antibacterial agents didn’t affect biofilm formation by E coli strain.2.2 Effects of CAZ and other drugs on biofilm formations by E coli clinical isolatesCAZ at 1/4 MICs could inhibit E coli biofilm formation by some isolates(7/52), while CFP at 1/4 MICs might induce E coli biofilm formation by some isolates(17/52). CTX and CTR had scarcely effects on E coli biofilms formations.Biofilm formation by E coli clinical isolates E42 may not only be inhibited by CAZ at 1/4 MIC, but also induced by CFP at 1/4 MIC.2.3 Dose-Effects and Time-Effects relationship of CAZ and CFP on biofilm formationStudies on dose-effect and time-effect relations showed that CAZ inhibited biofilm formations by ATCC700926 in a dose-dependent manner within the concentrations of 1/16-1/2 MICs, and that by E46 and E42 within the 1/8-1/2 MICs, while CFP may induce biofilm formation by E42 in a dose-dependent manner within the concentrations of 1/16-1/2 MICs. Effects of CFP on biofilm formation by E49 did not show a dose-dependent manner, and ceiling effect occurred at 1/4MIC. Time-effect analysis indicated that effects of sub-MIC of ceftazidime and cefoperazone on E coli biofilm may reach the maximum at3.1.2 Correlation test of ESBLs molecular and the effects of CAZ and CFPBiofilm formation by ATCC7000926 and E46 may both be inhibited by CAZ. While ATCC700926 carried no ESBL gene, and E46 carried TEM and CTX-M enzyme. Both E42 and E49 carried TEM, but their responses to drugs were not similar. These results indicated that the effects of CAZ and CFP were not correlated to ESBL genes they carried.3.2 Concentration analysis of CAZ and CFP on biofilm formation of E coliThe concentration of CAZ in inhibiting the biofilm was gradually decreased, and only 39.3% was remained in 6 hours in the presence of E46. The concentration of CFP in inducing the biofilm was completely degraded in 2 hours in the presence of E11.3.3 Measurement of mRNA changes of the genes encoding biofilm-modulating proteinsTo examine which regulator gene is involved in the effects of CAZ and CFP on E coli biofilm formation, we evaluated changes in the mRNA levels of important regulator genes, including QS associated genes, pfs, lux S and tnaA, and biofilm associated genes ariR, hha and tom B, in bacterial cultures 3 h after inoculation. Among these six genes, only levels of luxS mRNA were associated with CAZ or CFP treatment, suggesting that luxS/AI-2 QS could be involved in the inhibitory effects of CAZ and the inductive functions of CFP on E. coli biofilm formation.4. Mechanism of CAZ and CFP on E coli biofilm formation via lux S/AI-24.1 Influence of CAZ and CFP at 1/4 MICs on E coli luxS/AI-24.1.1 Influence of CAZ and CFP at 1/4 MICs on growth of E coli.CAZ and CFP did not affect the growth of the 4 strains of E coli, indicating that the biofilm influencing activities of CAZ and CFP at sub-MICs were not achieved through inhibiting or inducing the growth of bacteria.4.1.2 Influence of CAZ and CFP at 1/4 MICs on lux S mRNA, AI-2 production and m RNA of the genes modulated by AI-2We further simultaneously quantified mRNA levels of lux S and AI-2 production in the isolates E42, E46, E49 and ATCC700926 in the presence of 1/4 MICs of CAZ or CFP. Both CAZ and CFP at 1/4 MICs exerted effects on m RNA levels of lux S and AI-2 production. In E42, E46 and ATCC700926, m RNA levels of lux S were significantly less after growth in LB medium with 1/4 MIC CAZ, compared to the controls in which CAZ was absent. However, In E42 and E49, these levels were higher after exposure to 1/4 MIC CFP. Based on the AI-2 bioassay, changes in bioluminescence occurred after treatment for 4 h. These results agreed with those of mRNA levels of luxS.During biofilm formation, AI-2 stimulates biofilm formation and changes its architecture by stimulating flagellar motility via Mqs R, Qse BC and McbR. We measured the m RNA level of key downstream genes that are regulated by AI-2 in the cultures incubated with CAZ or CFP at 1/4 MICs for 12 h. The expression levels of mqsR, qseB, qse C, and mcbR in E42, E46, E49 and ATCC700926 were significantly less in the presence of 1/4 MIC of CAZ, whereas CFP exerted the opposite effect on these genes.4.2 Effects of as-ODNs targeting luxS on biofilm-modulating roles of CAZ and CFPTo confirm the roles of lux S in the responses to CAZ and CFP, we employed as-ODNs targeting lux S. AI-2 production was measured to verify whether lux S expression was blocked. Our results showed that the transduction of as-ODNs targeting lux S decreased AI-2 production significantly, indicating that lux S expression was successful blocked. Because as-ODN-4 was the most efficient at blocking lux S expression, we selected it for the subsequent experiments. We then quantified E. coli biofilm formation in the presence of 1/4 MIC of CAZ or CFP. After as-ODN transduction, the effects of CAZ and CFP on E. coli biofilm formation were diminished, suggesting that CAZ and CFP were unable to in fluence biofilm formation once the lux S gene was blocked.4.3 Effects of CAZ at 1/4MIC on biofilm formation by ATCC700926△lux SSub MIC CAZ does not influence biofilm formation by ATCC700926 △ luxS, indicating that CAZ and CFP influence E coli biofilm formation via lux S/AI-2 quorum sensing. In our previous study( see results 2.1), sub MIC CAZ does not influence biofilm formation by DH5α in which AI-2 could not be synthesized, which provides more evidence for the view.Conclusions:1. CAZ at 1/4 MICs can inhibit E coli biofilm formation by some isolates(7/52), while CFP at 1/4 MICs may induce E coli biofilm formation by some isolates(17/52).2. Biofilm formation by E coli clinical isolates E42 may not only be inhibited by CAZ at 1/4 MIC, but also induced by CFP at 1/4 MIC.3. Sub-MIC CAZ and CFP do not affect the growth of E coli strains, indicating that the biofilm influencing activities of CAZ and CFP at sub-MICs are not achieved through inhibiting or inducing the growth of bacteria, and the mechanisms of CAZ and CFP on E coli biofilm formation are completely different from their antibacterial mechanisms.4. Effects of CAZ and CFP at sub-MICs on E coli biofilm formation are not correlated with the drug resistance of bacteria.5. Sub-MIC CAZ and CFP influence E coli biofilm formation via lux S/AI-2 quorum sensing.